Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing

Chitosan, a well known natural cationic polysaccharide, has been successfully implemented in vitro and in vivo as a nonviral delivery system for both plasmid DNA and siRNA. While using chitosan/siRNA polyplexes to knock down specific targets, we have underestimated the effect of nucleic acids binding to chitosan when extracting RNA for subsequent quantitative PCR evaluation of silencing. In vitro transfection using chitosan/siRNA-based polyplexes reveals a very poor recovery of total RNA especially when using low cell numbers in 96 well plates. Here, we describe a method that dramatically enhances RNA extraction from chitosan/siRNA-treated cells by using an enzymatic treatment with a type III chitosanase. We show that chitosanase treatment prior to RNA extraction greatly enhances the yield and the integrity of extracted RNA. This method will therefore eliminate the bias associated with lower RNA yield and integrity when quantifying gene silencing of chitosan-based systems using quantitative real time PCR

Low molecular weight chitosan nanoparticulate system at low N:P ratio for nontoxic polynucleotide delivery

Chitosan, a natural polymer, is a promising system for the therapeutic delivery of both plasmid DNA and synthetic small interfering RNA. Reports attempting to identify the optimal parameters of chitosan for synthetic small interfering RNA delivery were inconclusive with high molecular weight at high amine-to-phosphate (N:P) ratios apparently required for efficient transfection. Here we show, for the first time, that low molecular weight chitosan (LMW-CS) formulations at low N:P ratios are suitable for the in vitro delivery of small interfering RNA. LMW-CS nanoparticles at low N:P ratios were positively charged (ζ-potential ~20 mV) with an average size below 100 nm as demonstrated by dynamic light scattering and environmental scanning electron microscopy, respectively. Nanoparticles were spherical, a shape promoting decreased cytotoxicity and enhanced cellular uptake. Nanoparticle stability was effective for at least 20 hours at N:P ratios above two in a slightly acidic pH of 6.5. At a higher basic pH of 8, these nanoparticles were unravelled due to chitosan neutralization, exposing their polynucleotide cargo. Cellular uptake ranged from 50% to 95% in six different cell lines as measured by cytometry. Increasing chitosan molecular weight improved nanoparticle stability as well as the ability of nanoparticles to protect the oligonucleotide cargo from nucleases at supraphysiological concentrations. The highest knockdown efficiency was obtained with the specific formulation 92-10-5 that combines sufficient nuclease protection with effective intracellular release. This system attained >70% knockdown of the messenger RNA, similar to commercially available lipoplexes, without apparent cytotoxicity. Contrary to previous reports, our data demonstrate that LMW-CS at low N:P ratios are efficient and nontoxic polynucleotide delivery systems capable of transfecting a plethora of cell lines

Stability and binding affinity of DNA/chitosan complexes by polyanion competition

The stability of DNA/chitosan complexes upon exposure to hyaluronic acid, chondroitin sulfate, and heparin, was assessed by fluorescence spectroscopy to quantify DNA release. Only the highly charged heparin was found to release DNA from the complexes. Complex stability upon exposure to heparin increased with the degree of deacetylation and molecular weight of chitosan and with the ratio of chitosan amino groups to DNA phosphate groups (N/P ratio) in the complexes. Isothermal titration microcalorimetry revealed that among polyanions tested, only heparin has a binding affinity to chitosan approaching that of DNA and can therefore release DNA from the complexes. These results also indicate that anionic components with sufficiently high charge density can induce extracellular or intracellular release of DNA, the former negatively affecting delivery efficiency while the latter is required for gene transfer to occur. Our findings also suggest that increased N/P ratio of the complexes can play an important role in preventing premature dissociation of DNA/polycation complexes upon interaction with anionic components in extracellular milieu. (C) 2017 Elsevier Ltd. All rights reserved.Peer reviewe

Differential protein profiling as a potential multi-marker approach for TSE diagnosis

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177This "proof of concept" study, examines the use of differential protein expression profiling using surface enhanced laser desorption and ionisationtime of flight mass spectrometry (SELDI-TOF) for the diagnosis of TSE disease. Spectral output from all proteins selectively captured from individual murine brain homogenate samples, are compared as "profiles" in groups of infected and non-infected animals. Differential protein expression between groups is thus highlighted and statistically significant protein "peaks" used to construct a panel of disease specific markers. Studies at both terminal stages of disease and throughout the time course of disease have shown a disease specific protein profile or "disease fingerprint" which could be used to distinguish between groups of TSE infected and uninfected animals at an early time point of disease. Results Our results show many differentially expressed proteins in diseased and control animals, some at early stages of disease. Three proteins identified by SELDI-TOF analysis were verified by immunohistochemistry in brain tissue sections. We demonstrate that by combining the most statistically significant changes in expression, a panel of markers can be constructed that can distinguish between TSE diseased and normal animals. Conclusion Differential protein expression profiling has the potential to be used for the detection of disease in TSE infected animals. Having established that a "training set" of potential markers can be constructed, more work would be required to further test the specificity and sensitivity of the assay in a "testing set". Based on these promising results, further studies are being performed using blood samples from infected sheep to assess the potential use of SELDI-TOF as a pre-mortem blood based diagnostic.https://doi.org/10.1186/1471-2334-9-1889pubpub

Enhanced Gene Delivery Mediated by Low Molecular Weight Chitosan/DNA Complexes: Effect of pH and Serum

This study was designed to systematically evaluate the influence of pH and serum on the transfection process of chitosan-DNA complexes, with the objective of maximizing their efficiency. The hydrodynamic diameter of the complexes, measured by dynamic light scattering (DLS), was found to increase with salt and pH from 243 nm in water to 1244 nm in PBS at pH 7.4 and aggregation in presence of 10% serum. The cellular uptake of complexes into HEK 293 cells assessed by flow cytometry and confocal fluorescent imaging was found to increase at lower pH and serum. Based on these data, new methodology were tested and high levels of transfection (>40%) were achieved when transfection was initiated at pH 6.5 with 10% serum for 8-24 h to maximize uptake and then the media was changed to pH 7.4 with 10% serum for an additional 24-40 h period. Cytotoxicity of chitosan/DNA complexes was also considerably lower than Lipofectamine. Our study demonstrates that the evaluation of the influence of important parameters in the methodology of transfection enables the understanding of crucial physicochemical and biological mechanisms which allows for the design of methodologies maximising transgene expression

Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria: kingdom Orthornavirae: phylum Negarnaviricota)

In April 2023, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV

3D morphometric analysis of calcified cartilage properties using micro-computed tomography

OBJECTIVE: Our aim is to establish methods for quantifying morphometric properties of calcified cartilage (CC) from micro-computed tomography (muCT). Furthermore, we evaluated the feasibility of these methods in investigating relationships between osteoarthritis (OA), tidemark surface morphology and open subchondral channels (OSCCs). METHOD: Samples (n = 15) used in this study were harvested from human lateral tibial plateau (n = 8). Conventional roughness and parameters assessing local 3-dimensional (3D) surface variations were used to quantify the surface morphology of the CC. Subchondral channel properties (percentage, density, size) were also calculated. As a reference, histological sections were evaluated using Histopathological osteoarthritis grading (OARSI) and thickness of CC and subchondral bone (SCB) was quantified. RESULTS: OARSI grade correlated with a decrease in local 3D variations of the tidemark surface (amount of different surface patterns (rs = -0.600, P = 0.018), entropy of patterns (EP) (rs = -0.648, P = 0.018), homogeneity index (HI) (rs = 0.555, P = 0.032)) and tidemark roughness (TMR) (rs = -0.579, P = 0.024). Amount of different patterns (ADP) and EP associated with channel area fraction (CAF) (rp = 0.876, P < 0.0001; rp = 0.665, P = 0.007, respectively) and channel density (CD) (rp = 0.680, P = 0.011; rp = 0.582, P = 0.023, respectively). TMR was associated with CAF (rp = 0.926, P < 0.0001) and average channel size (rp = 0.574, P = 0.025). CC topography differed statistically significantly in early OA vs healthy samples. CONCLUSION: We introduced a mu-CT image method to quantify 3D CC topography and perforations through CC. CC topography was associated with OARSI grade and OSCC properties; this suggests that the established methods can detect topographical changes in tidemark and CC perforations associated with OA

Molecular, Biochemical and Genetic Characteristics of BSE in Canada

The epidemiology and possibly the etiology of bovine spongiform encephalopathy (BSE) have recently been recognized to be heterogeneous. In particular, three types [classical (C) and two atypical (H, L)] have been identified, largely on the basis of characteristics of the proteinase K (PK)-resistant core of the misfolded prion protein associated with the disease (PrPres). The present study was conducted to characterize the 17 Canadian BSE cases which occurred prior to November 2009 based on the molecular and biochemical properties of their PrPres, including immunoreactivity, molecular weight, glycoform profile and relative PK sensitivity. Two cases exhibited molecular weight and glycoform profiles similar to those of previously reported atypical cases, one corresponding to H-type BSE (case 6) and the other to L-type BSE (case 11). All other cases were classified as C-type. PK digestion under mild and stringent conditions revealed a reduced protease resistance in both of these cases compared to the C-type cases. With Western immunoblotting, N-terminal-specific antibodies bound to PrPres from case 6 but not to that from case 11 or C-type cases. C-terminal-specific antibodies revealed a shift in the glycoform profile and detected a fourth protein fragment in case 6, indicative of two PrPres subpopulations in H-type BSE. No mutations suggesting a genetic etiology were found in any of the 17 animals by sequencing the full PrP-coding sequence in exon 3 of the PRNP gene. Thus, each of the three known BSE types have been confirmed in Canadian cattle and show molecular characteristics highly similar to those of classical and atypical BSE cases described from Europe, Japan and the USA. The occurrence of atypical cases of BSE in countries such as Canada with low BSE prevalence and transmission risk argues for the occurrence of sporadic forms of BSE worldwide